Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD) in the world and affects about 15-25% of type I diabetes patients and 30-40% of patients with type II diabetes. The development of DN is thought to result from the cumulative interactions among multiple metabolic and hemodynamic factors which activate intracellular signaling pathways that trigger the production of cytokines and growth factors leading to renal disease that progresses to glomerulosclerosis and interstitial sclerosis. Two of the most important growth factors driving fibrosis in DN are transforming growth factor-b (TGFb) and connective tissue growth factor (CTGF), which is induced by TGFb. Therapeutic strategies to inhibit the activity of TGFb and CTGF have been proposed to halt the progression of DN. This proposal will develop a novel chimeric Fc fusion protein designed to inhibit the pro-fibrotic activities of both TGFb and CTGF. CCN3/HGF is a heterodimeric fusion protein consisting of the CTGF antagonist, CCN3, and the alpha chain fragment of hepatocyte growth factor (HGF) both fused to the N-terminus if the Fc portion of IgG. Heterodimerization of the protein during mammalian cell expression is enforced by the use of the knobs into holes mutations in the Fc chains. The design of the Fc fusion protein is predicted to enhance stability and improve pharmacokinetics as shown for other Fc fusion proteins in clinical use. CCN3/HGF was shown to block TGFb and CTGF-induced pro-fibrotic responses in vitro in various types of kidney cells involved in the pathogenesis of DN fibrosis including fibroblasts, renal epithelial tubule cells, and mesangial cells. The goal of this project is to advance the preclinical development of CCN3/HGF. Stable CHO cell lines producing high levels of CCN3/HGF will be developed. The anti-fibrotic activity of the fusion protein will be confirmed in vitro using differnt kidney cell types. Therapeutic efficacy of CCN3/HGF will be evaluated in the eNOS-/-/leprdb/db double-knockout mouse model of DN. The results of these studies will pave the way for submission of an IND in future work. The unique dual mechanism of action of CCN3/HGF will provide a new therapeutic option potentially superior to the relatively limited and ineffective current treatments for DN patients.